ES2958621T3 - Compositions for the topical treatment of microbial infections - Google Patents

Abstract

The present invention provides compositions and methods for the topical treatment of infections. The compositions comprise glycerol monolaurate or a derivative thereof and are administered topically, for example, to treat viral, fungal or bacterial infections. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Compositions for the topical treatment of microbial infections

Cross reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 61/636,203, filed April 20, 2012, and U.S. Provisional Application No. 61/650,755, filed May 23, 2012. 2012.

Background of the invention

Some bacterial pathogens initiate disease in humans from intact or damaged mucosal or skin surfaces. Many of these pathogens are acquired from other people or animals, from endogenous sources, or from a myriad of environmental sources. Once in humans, pathogens colonize surfaces primarily as organismal biofilms, defined as thin films of organisms attached to host tissues, medical devices, and other bacteria through complex networks of polysaccharides, proteins, and nucleic acids. These bacteria can also exist as planktonic cultures (broths) in some host tissue environments, such as the bloodstream and mucosal secretions. Similarly, these potential pathogens can exist as biofilms or planktonic cultures in a myriad of non-living environments.

Glycerol monolaurate (GML) is a natural glycerol-based compound that has previously been shown to have antimicrobial, antiviral, and anti-inflammatory properties. The present invention provides GML compositions and methods for the treatment of various microbial infections and diseases resulting from one or more microbial infections.

Loo Chew Hung et al., "Testing of glyceryl monoesters for their anti-microbial susceptibility and their influence in emulsions", Journal of Oil Palm Research, 2010, describe a topical composition comprising monolaurin, hydrogenated palm kernel glyceride oil, palmitate isopropyl and Tween 80 for use in the treatment of Staphylococcus aureus and Aspergillus niger.

US 5256405A describes a deodorant stick with antibacterial properties comprising glyceryl monolaurate, coriander oil and a pharmaceutically acceptable optical carrier.

Kristmundsdottir et al., "Development and evaluation of microbial hydrogels containing monoglyceride as the active ingredient", 1999, describe microbial hydrogels containing various monoglycerides of lauric acid and capric acid to prevent the transmission of bacteria and viruses associated with sexually transmitted diseases.

Gill et al., "Evaluation of antilisterial action of cilantro oil on vacuum packed ham", International Journal of Food Microbiology, 2002, discloses that cilantro oil is not a suitable agent for the control of L. monocytogenese in ham.

"Preservation of products with surfactants" in Karabar and Orth "Preservative-free and self-preserving cosmetics and drugs - Principle and Practice", 1997, discloses the antibacterial activity of the combination of GML and EDTA.

Summary of the invention and disclosure

The invention is as set forth in the attached set of claims.

Any reference in the description to treatment methods refers to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for the treatment of the human (or animal) body by therapy (or for diagnosis).

Simple, inexpensive and well-tolerated methods and compositions are needed to apply antimicrobial compounds, such as GML, at effective levels to the skin and mucosal surfaces of humans and other vertebrates. The present invention addresses this and other needs.

In one aspect, the present disclosure is directed to a composition comprising glycerol monolaurate (GML) and a vegetable oil. In one embodiment, the vegetable oil is palm, olive, corn, canola, coconut, soybean or wheat, or a combination thereof. In a further embodiment, the vegetable oil is present in the composition at about 10% to about 99%, about 20% to about 90%, about 30% to about 80%, or about 40% to about 70%. In one embodiment, the composition comprising GML and a vegetable oil further comprises a pharmaceutically acceptable topical carrier, for example, petroleum jelly. In one embodiment, the GML is present in the composition at a concentration of about 10 μg/mL to about 100 mg/mL, from about 50 μg/mL to about 50 mg/mL, from about 100 μg/mL to about 10 mg. /mL, or from about 500 μg/mL to about 5 mg/mL. In another embodiment, the composition comprising GML and a vegetable oil further comprises a cellulose derivative, for example hydroxypropyl cellulose or hydroxyethyl cellulose, or a combination thereof. In a further embodiment, the cellulose derivative is present in the composition up to 1.25% w/w.

In another aspect, the present disclosure is directed to a composition comprising GML and a non-aqueous gel. In one embodiment, the composition comprising GML and a nonaqueous gel has a pH of about 4.0 to about 4.5. In one embodiment, the non-aqueous gel comprises polyethylene glycol, hydroxypropyl cellulose, hydroxyethyl cellulose or a combination thereof. In a further embodiment, the polyethylene glycol is present at approximately 25% w/w in the composition. In one embodiment, hydroxypropyl cellulose and hydroxyethyl cellulose are both present in the composition, each at a concentration of about 1.25% w/w.

In one embodiment, the GML composition comprising a nonaqueous gel comprises polyethylene glycol with a molecular weight range of about 300 to about 4000. In a further embodiment, the polyethylene glycol has a molecular weight of about 400 or about 1000.

In one embodiment, the GML composition comprising a non-aqueous gel further comprises a topical carrier, e.g. e.g. Vaseline. In a further embodiment, the composition comprises a vegetable oil.

In one embodiment, the compositions described herein comprise GML in a concentration of about 0.001% (w/v) to about 10% (w/v) of the total composition. In a further embodiment, the GML is present at about 0.005% (w/v) to about 5% (w/v) of the composition. In a further embodiment, the GML is present at about 0.01 to about 1%. In yet another embodiment, the GML is present at about 0.1% (w/v) to about 0.5% (w/v) of the composition.

In one embodiment, the GML is present in the composition at a concentration of about 10 μg/mL to about 100 mg/mL. In a further embodiment, the GML comprises about 50 μg/mL to about 50 mg/mL of the composition. In a further embodiment, the GML comprises about 100 μg/mL to about 10 mg/mL. In yet another embodiment, the GML comprises about 500 μg/mL to about 5 mg/mL.

In one embodiment, the GML composition provided herein comprises propylene glycol in a concentration of about 65% (w/w) to about 80% (w/w). In another embodiment, the polyethylene glycol is present in the composition in a concentration of about 20% (w/w) to about 35% (w/w). In one embodiment, both propylene glycol and polyethylene glycol are present in the topical composition.

In one embodiment, the composition comprises a cellulose derivative. In a further embodiment, the composition comprises hydroxypropyl cellulose or hydroxyethyl cellulose. In yet another embodiment, the cellulose is present in a concentration of about 0.1% (w/w) to about 5.0% (w/w).

In one embodiment, the GML composition comprises an aqueous solvent. In a further embodiment, the aqueous solvent is water, a saline solution, media or a combination thereof.

In one embodiment, the pharmaceutically acceptable topical carrier is petroleum jelly.

In one embodiment, the pH of the GML composition provided herein is about 4.0 to about 5.5.

In some embodiments, the composition provided herein comprises one or more accelerators. In a further embodiment, the accelerant is an organic acid, a chelator, an antibacterial agent, an antifungal agent, an antiviral agent or a combination thereof. In a further embodiment, the accelerant is a chelator. In yet a further embodiment, the accelerant is EDTA.

In another aspect, the GML composition provided herein has antimicrobial, antiviral and/or anti-inflammatory activity. For example, in one embodiment, the composition provided herein is applied topically to humans and other vertebrates, for example for the treatment of a bacterial, fungal or viral infection such as Gardnerella vaginalis or Candida albicans.

Accordingly, in one embodiment, the present disclosure provides methods for treating a microbial infection in a subject in need thereof. In one embodiment, the method comprises topically administering to the subject in need thereof an effective amount of a GML composition provided herein. In one embodiment, the composition comprises GML, a vegetable oil and a pharmaceutically acceptable topical carrier. In another embodiment, the composition comprises GML, a non-aqueous gel, and a pharmaceutically acceptable topical carrier. In a further embodiment, the composition comprises GML, a vegetable oil, a non-aqueous gel and a pharmaceutically acceptable topical carrier.

In one embodiment, the compositions described herein are applied topically with the use of a sponge, washcloth or swab.

In one embodiment, the subject has a bacterial infection. In a further embodiment, the bacterial infection is by Staphylococcus (such as Staphylococcus aureus); by Streptococcus (such as by Streptococcus pneumonia or Streptococcus agalactiae); by Escherichia (such as by Escherichia coli); Gardnerella (such as by Gardnerella vaginalis); Clostridium (such as by Clostridium perfringens); Mycobacterium (such as by Mycobacterium tuberculosis or Mycobacterium phlei); oChlamydia (such as Chlamydia trachomatis).

In another embodiment, the subject treated with one of the GML compositions provided herein has a fungal infection. In a further embodiment, the fungal infection is by Candida (such as Candida albicans), Microsporum species, Trichophyton species, Penicillium species, or Aspergillus species.

In another embodiment, the method of the disclosure involves administering a second active agent selected from the group consisting of antifungal agents, antiviral agents and antibiotics.

Brief description of the figures

Figure 1 is a graph showing the effect of various concentrations of GML in olive oil on the growth of various microorganisms (measured as CFU/mL). The bars represent the following microorganisms, from the left: Staphylococcus aureusMNPE (methicillin-sensitive strain), S.aureusMW2 (methicillin-resistant strain), Candida albicans, Streptococcus agalactiae, and Gardnerella vaginalis.

Figure 2 is a graph showing the effect of various concentrations of GML in palm oil on the growth of various microorganisms (measured as CFU/mL). The bars represent the following microorganisms, from the left: Staphylococcus aureusMNPE (methicillin-sensitive strain), S.aureusMW2 (methicillin-resistant strain), Candida albicans, Streptococcus agalactiae, and Gardnerella vaginalis.

Figure 3 is a graph showing the effect of various concentrations of GML in corn oil on the growth of various microorganisms (measured as CFU/mL). The bars represent the following microorganisms, from the left: Staphylococcus aureusMNPE (methicillin-sensitive strain), S.aureusMW2 (methicillin-resistant strain), Candida albicans, Streptococcus agalactiae, and Gardnerella vaginalis.

Figure 4 is a set of scanning electron micrographs showing S.aureus growing as a biofilm on buffer fibers in cellulose acetate dialysis tubes at 6000x (left) or 9000x (right) magnification.

Figure 5 is a graph showing the accumulation of TSST-1 superantigen in biofilms grown on buffer fibers in cellulose acetate dialysis tubes.

Figure 6 is a series of graphs showing the measured CFU/mL of biofilms formed from S.aureus strains MN8 (methicillin-sensitive strain, top panels), MNWH (methicillin-resistant strain, middle panels), and MW2 (methicillin-resistant strain, lower panels) cultured in 96-well plastic microtiter plates, in the presence or absence of the indicated concentrations of GML for 24 or 48 hours.

Figure 7 is a series of graphs showing biofilm absorbance measured at 595 nm after staining of biofilms with crystal violet, formed from S.aureusMN8 strains (methicillin-sensitive strain, upper panels). , MNWH (methicillin-resistant strain, middle panels) and MW2 (methicillin-resistant strain, bottom panels) cultured in 96-well plastic microtiter plates, for 24 or 48 hours, in the presence or absence of the indicated concentration of GML.

Figure 8 is a set of graphs showing CFU/mL (top) and absorbance at 595 nm measured after crystal violet staining (bottom) of Haemophilus influenza biofilms cultured in 96-well plastic microtiter plates, for 24 or 48 hours, in the presence or absence of the indicated concentration of GML.

Figure 9 is a set of graphs showing CFU/mL (left) and absorbance at 595 nm measured after crystal violet staining (right) of S.aureusoH influenza biofilms treated with 500 μg/mL GML.

Figure 10 is a graph showing CFU/mL of cultures of E. colicultured for 24 hours in the presence or absence of 100 μg/mL GML and in the presence of increasing concentrations of EDTA.

Figure 11 is a graph showing CFU/mL of cultures of E. colicultured for 24 hours in 100 μg/mL GML alone, increasing concentrations of EDTA alone, or increasing concentrations of EDTA in the presence of 100 μg/mL GML.

Figure 12 is a graph showing the CFU/mL of S.aureus cultures grown at the indicated pH and the indicated concentrations of GML.

Figure 13 is a set of graphs showing the CFU/mL of H influenza cultures grown at the indicated pH, in the presence of 1 μg/mL of GML.

Figure 14 is a graph showing CFU/mL of Pseudomonas aeruginosa cultures grown at the indicated pH and indicated GML concentrations.

Figure 15 is a graph showing the effect of the indicated concentrations of GML alone (black), GML in a 10% non-aqueous carrier gel (white) or a 25% non-aqueous carrier gel (gray), on the S.aureus growth (CFU/mL).

Figure 16 is a graph depicting the solubility of tenofovir (10 mg/mL) in GML at a pH ranging from 4.0 to 4.5. The absence of a bar indicates that tenofovir at a concentration of 10 mg/mL was not soluble in the composition, while the presence of a bar indicates that tenofovir at a concentration of 10 mg/mL was soluble in the composition.

Figure 17 is a set of graphs showing the bactericidal activity of the indicated forms of GML against S.pyogenes.

Figure 18 is a graph showing the bactericidal activity (CFU/mL) of GML compared to lauric acid, in the presence of S.pyogenes.

Figure 19 is a graph showing the bactericidal activity (CFU/mL) of GML compared to lauric acid, in the presence of S.aureus.

Figure 20 is a graph showing the production of superantigen from S.aureus(TSST-1) or S.pyogenes(SPE A) in the presence of g Ml or lauric acid.

Figure 21 is a graph showing the bactericidal activity of GML after pretreatment with S.aureus or S.pyogenes.

Figure 22 is a graph showing staphylococcal counts (CFU/mL) in anterior nares of three human subjects treated with 5% GML in a non-aqueous gel.

Figure 23 is a graph showing the ability of 5% GML in a non-aqueous gel to reduce aerobic bacteria on human teeth and gums (CFU/mL).

Figure 24 is a graph showing the presence of S.aureus in the surgical incision sites of rabbits 24 hours after cleaning with S.aureusMN8 and PBS or 5% GML

Figure 25 shows the inflammation present at surgical incision sites of New Zealand white rabbits treated with S.aureusMN8 and then GML gel (left) or PBS (right) for 24 hours. Inflammation manifests as a dark gray discoloration of the surgical site, as indicated by the arrows.

Detailed description of the invention and disclosure

The present disclosure provides topical GML compositions and methods of treatment with the compositions, e.g. e.g., by topical administration. The compositions and methods provided herein, in one embodiment, are used to treat infections topically, for example, by facilitating the administration of effective amounts of GML to the skin or a mucosal surface of a subject, for example, a human being. Without wishing to be bound by theory, it is believed that the compositions of the invention result in greater patient compliance with topical self-administration due to the less irritating nature of the composition, relative to previously employed topical formulations of antimicrobial compounds. and antivirals.

As used herein, the term "antimicrobial" means effective in preventing, inhibiting or arresting the growth or pathogenic effects of a microorganism. "Microorganism" is used herein to refer to any bacteria, virus or fungus. In one embodiment, the formulations of the disclosure are used to prevent, inhibit or stop the growth of one or more of the following microorganisms: Staphylococcus aureus, Streptococcus (e.g., S.pyogenes, S. agalacticae or S, or S. . pneumoniae) Haemophilus influenzae, Pseudomonas aeruginosa, Gardnerella vaginalis, Enterobacteriacae (e.g. Escherichia coli), Clostridium perfringens, Chlamydia trachomatis, Candida albicans, Human Immunodeficiency Virus (HIV) or Herpes Simplex Virus (HSV).

"Antibacterial" or "antifungal", as used herein, refers to the inhibition or arrest of the growth of a bacteria or fungus, a reduction in the severity or likelihood of developing a bacterial or fungal disease, which induces death of the bacteria or fungus or reduction or inhibition of the pathogenic effects of the respective bacteria or fungus. "Bactericidal" is used interchangeably with "antibacterial."

"Antiviral", as used herein, refers to the inhibition of viral infection or virus replication, a reduction in the likelihood that a subject exposed to a virus will contract the viral disease, or a reduction in the severity of the viral disease.

The term "effective amount", as used herein, refers to an amount that is sufficient to effect a beneficial or desired antimicrobial activity, including, without limitation, killing the microorganism or inhibiting infection, growth or toxicity. microbial. An effective amount of GML is about 10 μg/mL, about 100 μg/mL, about 1 mg/mL, about 10 mg/mL, about 50 mg/mL, or about 100 mg/mL.

The terms "treat", "treatment" and "treating" refer to an approach to obtain beneficial or desired results, for example, clinical results. For the purposes of this invention, beneficial or desired results may include inhibiting or suppressing the growth of a microorganism or killing a microorganism; inhibit one or more processes by which a microorganism infects a cell or subject; inhibit or ameliorate the disease or condition caused by microbial infection; or a combination thereof. The terms "treat", "treatment" or "that treats" also refer to the prophylaxis of infection. In some embodiments, the formulations of the disclosure are used to treat urinary tract infections, vaginal microbial infections, oral cavity infections such as those that cause gum disease, postsurgical infections including respiratory tract infections, wound infections or of the surgical incision site, or infections characterized by the production of toxins, including toxic shock syndrome.

"Prophylaxis" as used herein may mean the complete prevention of an infection or disease, or the prevention of the development of symptoms of that infection or disease; a delay in the onset of an infection or disease or its symptoms; or a decrease in the severity of a subsequently developed infection or disease or its symptoms.

As used herein, the term "subject" includes humans and other animals. The subject, in one embodiment, is a human being.

"Topical", as used herein, refers to the application of the composition to any surface of the skin or mucosa. "Skin surface" refers to the protective outer covering of a vertebrate's body, generally comprising a layer of epidermal cells and a layer of dermal cells. A “mucosal surface,” as used herein, refers to a tissue lining of an organ or body cavity that secretes mucosa, including, but not limited to, the oral, vaginal, rectal, gastrointestinal, and nasal surfaces. In one embodiment, the formulations of the invention are administered topically to the areas of the teeth and gums, the skin, the nose or the vagina.

The term "pharmaceutically acceptable topical carrier," as used herein, refers to a material, diluent or carrier that can be applied to the skin or mucosal surfaces without undue toxicity, irritation or allergic reaction.

A "pharmaceutically acceptable excipient" means an excipient that is useful for preparing a pharmaceutical composition that is generally safe, non-toxic and not biologically or otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as for human pharmaceutical use. A "pharmaceutically acceptable excipient" as used in the present application includes both one and more than one such excipient.

As used herein, the term "vegetable oil" means a substance extracted from a plant or seed that exists in liquid form at room temperature. Suitable vegetable oils include, but are not limited to, palm, olive, corn, canola, coconut, soybean, wheat germ, jojoba, sunflower, sesame, peanut, cottonseed, safflower, soybean, rapeseed, almond, beech, cashew, hazelnut , macadamia, mongongo nut, pecan, pine nut, pistachio, walnut, grapefruit seed, lemon, orange, bitter gourd, bottle gourd, buffalo gourd, pumpkin seed, egusi seed, pumpkin seed, watermelon seed, acai, black seed, black currant seed, orange seed, evening primrose, linseed, eucalyptus, amaranth, apricot, apple seed, argan, avocado, babassu, coriander seed, grape seed, mustard, poppy seed, bran rice, castor, or mixtures thereof. The mixtures may be, by way of example and without limitation, a combination of olive oil and soybean oil, a combination of coconut oil and wheat germ oil, or a combination of jojoba oil, palm oil and oil. of castor. Vegetable oil mixtures can be binary, ternary, quaternary or higher mixtures.

The term "accelerant", as used herein, refers to a compound, substance, liquid, powder or mixture that, when added to the composition, has the effect of enhancing or contributing to the antimicrobial properties of the composition. . The accelerants may be an organic acid including, but not limited to, lactic acid, ascorbic acid, citric acid, formic acid, benzoic acid and oxalic acid. The accelerant, in another embodiment, is a chelator, and in one embodiment, it is selected from ethylenediaminetetraacetic acid (EDTA), dimercaprol, dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), alpha- lipoic acid (ALA), or combinations thereof. In another embodiment, the accelerant is selected from an antibiotic agent, an antifungal agent, an antiviral agent or a combination thereof. Antibiotics for use with the invention, for example, include aminoglycosides, carbacephemes, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, penicillins, polypeptides, quinolones, sulfuramides and tetracyclines. Antifungal agents include, but are not limited to, those of the azole class, polyenes or echinocanins, nucleoside analogues, allylamines, griseofulvin, tolnaftate or selenium compounds. Antiviral agents include, for example and without limitation, acyclovir, ganciclovir, valganciclovir, abacavir, enofovir, lamivudine, emtricitabine, zidovudine, tenofovir, efavirenz, raltegravir, enfuvirmide, maraviroc, ribavirin, amantadine, rimantadine, interferon, oseltamivir and zanamivir.

As used herein, the term "cellulose derivative" refers to any cellulose-based compound and may include, for example, hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose or cellulose acetate.

The term "biofilm", as used herein, means an aggregate of microorganisms, generally bacterial, adhered to each other and growing on a surface. Microbial cells in the biofilm typically produce an extracellular matrix known as extracellular polymeric substance. Often, this matrix and the density of the aggregate itself significantly increase the antibiotic resistance of bacteria in the biofilm. Biofilms may be involved in urinary tract infections, oral infections, and dental diseases such as gingivitis, and may also form on the surface of implanted devices, including prostheses, catheters, or heart valves.

In one aspect, the present disclosure provides a topical composition comprising glycerol monolaurate (GML). In a further embodiment, the composition comprises a vegetable oil or a non-aqueous gel, or a combination thereof. The non-aqueous gel, in one embodiment, comprises a cellulose derivative. The topical composition provided herein, in one embodiment, comprises a pharmaceutically acceptable topical carrier.

In one embodiment, the composition provided herein comprises the monoglyceride GML. GML is a fatty acid ester of glycerol, derived from lauric acid, with the chemical formula C<15>H<30>O<4>. GML is also known in the art as glyceryl laurate or monolaurin. GML is found naturally in breast milk and some plants, and is used as a food and cosmetic additive. GML and other glycerides are listed in the US Food and Drug Administration's Substances Generally Recognized as Safe database. GML and related compounds have been previously described in US Patent Application Nos. 10 /579,108 (filed on November 10, 2004) and 11/195,239 (filed on August 2, 2005).

GML can be synthesized in multiple forms, including the R and S optical isomers, as well as forms with lauric acid in the 1/3 positions and in the 2 position. The composition provided herein, in one embodiment, comprises the isomer R for GML. In another embodiment, the composition provided herein comprises the S isomer of GML. In yet another embodiment, a racemic mixture of isomers is provided in the composition.

Similarly, the topical composition may comprise GML with lauric acid at positions 1/3, GML with lauric acid at position 2, or a combination thereof. The R and S isomers of each form, and their racemic mixtures, are susceptible of use with the present invention.

The chemical structure of GML with lauric acid in the 1/3 positions is

Glycerol monolaurate (GML) in positions 1/3

The chemical structure of GML with lauric acid in position 2 is:

Disclosed herein is a derivative of GML, for example a compound selected from one of Formulas I-VI. Examples of such compounds include, by way of example and without limitation, glycerol monocaprylate, glycerol monocaprate, glycerol monomyristate, glycerol monopalmitate and dodecylglycerol.

Where each occurrence of X is independently -O- or -S-; and n is an integer from 5 to 20 (inclusive). Also disclosed is a derivative of GML, and said at least one derivative is a compound of Formula V or Formula VI. Examples of such compounds include, but are not limited to, glycerol dilaurate, glycerol dicaprylate, glycerol dimyristate, glycerol trilaurate and glycerol tripalmitate.

Where each occurrence of X is independently -O- or -S-; and each occurrence of n is independently an integer from 5 to 20 (inclusive).

In one embodiment, a compound of Formula I, II, III or IV is present in the topical composition of the disclosure, and at least one -X- is -S-. In one embodiment, one occurrence of -X- is -S- and the remaining occurrences of -X- are -O-. In one embodiment, a compound of Formula V or VI is present in the topical composition of the disclosure, each occurrence of n is 10 and at least one -X- is -O-.

The topical composition provided herein, in one embodiment, comprises GML and a derivative of GML. For example, in one embodiment, the topical composition provided herein comprises GML and a compound of Formula VI. In a further embodiment, each occurrence of n is 10 and at least one -X- is -O-. In one embodiment, the topical composition comprises GML from about 0.001% (w/v) to about 10% (w/v) of the composition. In a further embodiment, the GML comprises about 0.005% (w/v) to about 5% (w/v) of the composition. In yet another embodiment, the GML comprises about 0.01% (w/v) to about 1.0% (w/v) of the composition. In yet a further embodiment, the GML or a derivative thereof comprises about 0.05% (w/v) to about 0.5% (w/v) of the composition.

In another embodiment, the topical composition comprises GML in a concentration of about 10 μg/mL to about 100 mg/mL. In a further embodiment, the topical composition comprises GML in a concentration of about 50 μg/mL to about 50 mg/mL. In a further embodiment, the topical composition comprises GML in a concentration of about 100 μg/mL to about 10 mg/mL. In yet a further embodiment, the topical composition comprises GML in a concentration of about 500 μg/mL to about 5 mg/mL.

In one embodiment, the topical composition comprises GML in a concentration of about 10 μg/mL, about 50 μg/mL, about 100 μg/mL, about 500 μg/mL, about 1 mg/mL, about 5 mg/mL, about 10 mg/mL, approximately 50 mg/mL or approximately 100 mg/mL. The amount of GML in the composition can be adapted according to the condition/disease being treated as well as the characteristics of the subject being treated. The amount of GML in the composition may vary depending, for example, on the nature of the infection or disease; the administration site; the subject's medical history, weight, age, sex and treated area; and if the subject is receiving any other medications.

As provided above, in one aspect, the present disclosure is directed to a topical composition comprising GML.

In one embodiment, the topical composition comprises at least one glycol. For example, in one embodiment, the topical composition comprises propylene glycol, polyethylene glycol, or a combination thereof. In one embodiment, the polyethylene glycol has a molecular weight (MW) in the range of about 300 to about 10,000. In a further embodiment, the polyethylene glycol has a molecular weight of about 300 to about 1,000. In yet another embodiment, the polyethylene glycol has a molecular weight of about 400.

In one embodiment, polyethylene glycol is present in the topical composition. In a further embodiment, the polyethylene glycol has a MW of about 400, about 500, or about 1,000. In one embodiment, the polyethylene glycol is present in the topical composition in a concentration (w/w) of about 15% to about 50%, about 20% to about 40%, or about 25% to about 35%, for example, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50%. In a further embodiment, both propylene glycol and polyethylene glycol are present in the topical composition. In a further embodiment, the propylene glycol is present at a concentration of about 70% to about 80% and the polyethylene glycol is present at a concentration of about 20% to about 30%. In yet a further embodiment, the polyethylene glycol is polyethylene glycol 400.

In another embodiment, a topical composition comprising GML is provided. In a further embodiment, propylene glycol is present in the composition. In yet a further embodiment, the propylene glycol is present in the composition in a concentration of about 60% to about 80%, e.g., about 60%, about 65%, about 70%, about 71%, about 72%, about 73 %%, approximately 74%, approximately 75% or approximately 80%.

In another embodiment, a topical composition comprising GML is provided. In one embodiment, the topical composition comprises at least one cellulose derivative. In a further embodiment, the composition comprises one or two cellulose derivatives. In one embodiment, the cellulose derivative is hydroxypropyl cellulose. In another embodiment, the cellulose derivative is hydroxyethylcellulose, carboxymethylcellulose or hydroxymethylcellulose. In yet another embodiment, the composition comprises a combination of hydroxyethylcellulose and hydroxypropylcellulose. In one embodiment, the cellulose derivative is present in a concentration of about 0.1% (w/w) to about 5.0% (w/w). In a further embodiment, multiple cellulose derivatives are present in the composition at the same concentration. In a further embodiment, two cellulose derivatives are present, and each is present at a concentration of about 1.25% (w/w). Cellulose derivatives include, for example, hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose or cellulose acetate.

In one embodiment, the topical composition provided herein comprises GML, at least one cellulose derivative, propylene glycol and polyethylene glycol.

In another embodiment, a topical composition comprising GML is provided. In a further embodiment, the composition comprises at least one vegetable oil, for example, at least one of the vegetable oils described above (e.g., palm oil, olive oil, corn oil). In one embodiment, the vegetable oil is present in the composition in a concentration of about 0.1% (w/w) to about 10% (w/w). In a further embodiment, the vegetable oil is present in the composition in a concentration of about 1% (w/w) to about 8% (w/w). In a further embodiment, the vegetable oil is present in the composition in a concentration of about 1% (w/w) to about 6% (w/w). In a further embodiment, the vegetable oil is present in the composition in a concentration of about 1% (w/w) to about 4% (w/w). In one embodiment, the vegetable oil is present in the composition in a concentration of about 0.1% (w/w), about 0.5% (w/w), about 1.0% (w/w), about 1.25% (w/w), approximately 1.5% (w/w), approximately 1.75% (w/w) or approximately 2.0% (w/w).

In one embodiment, the topical composition provided herein comprises a vegetable oil and at least one cellulose derivative. For example, in one embodiment, the topical composition comprises hydroxypropyl cellulose and a vegetable oil, or hydroxyethyl cellulose and a vegetable oil, or a combination of hydroxypropyl cellulose, hydroxyethyl cellulose and a vegetable oil. In one embodiment, the cellulose derivative and the vegetable oil (e.g., palm oil or corn oil) are each present at the same concentration (w/w). In a further embodiment, the cellulose derivative and the vegetable oil are each present in the composition at about 1% (w/w) to about 5% (w/w). In yet a further embodiment, the cellulose derivative is a combination of hydroxypropyl cellulose and hydroxyethyl cellulose, and each is present in the composition at about 1.25% (w/w). In one embodiment, the composition comprises a vegetable oil and two cellulose derivatives. In a further embodiment, the two cellulose derivatives are hydroxypropyl cellulose and hydroxyethyl cellulose, and the total concentration of cellulose derivatives in the composition is approximately 1.25% (w/w). Cellulose derivatives include, for example, hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose or cellulose acetate.

In some embodiments, the topical composition provided herein comprises one or more accelerants. In a further embodiment, the accelerant is an organic acid, a chelator, an antibacterial agent, an antifungal agent, an antiviral agent or a combination thereof. In a further embodiment, the accelerant is a chelator. In yet a further embodiment, the accelerant is EDTA.

The accelerant, in one embodiment, is EDTA. In a further embodiment, the GML composition provided herein comprises EDTA in a concentration of about 0.00005 M, about 0.0005 M, about 0.005 or about 0.05 M. In another embodiment, a chelator is present in the composition in a concentration of about 0.00005 M to about 0.05 M, about 0.0005 M to about 0.005 M, or about 0.005 to about 0.05 M.

In one embodiment, the topical composition comprises both a vegetable oil and an accelerant, for example palm oil and EDTA. In another embodiment, the accelerant is an organic acid and is present in the formulation with a vegetable oil. In one embodiment, the topical composition provided herein comprises an accelerator and a nonaqueous gel, for example a gel comprising a cellulose derivative. In another embodiment, the topical composition comprises GML or a derivative thereof, a vegetable oil, a non-aqueous gel (e.g., a gel comprising one or more cellulose derivatives), and an accelerator.

In one embodiment, the composition contains at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well known to those skilled in the art and may include buffers (e.g., phosphate buffer and citrate buffer), amino acids, alcohols, proteins such as serum albumin, parabens (e.g., methylparaben), or mannitol.

The pH of the composition is about 4.0 to about 4.5.

In one embodiment, the composition provided herein comprises GML and a pharmaceutically acceptable topical carrier. In one embodiment, the pharmaceutically acceptable topical carrier is a mixture of hydrocarbons such as, for example, paraffin wax or petroleum jelly. Vaseline is any semi-solid, hydrophobic, water-insoluble mixture of hydrocarbons. The pharmaceutically acceptable topical carrier can be added to any of the formulations described herein.

In another embodiment, the composition comprises an aqueous solvent. Compositions comprising an aqueous solvent may or may not include a pharmaceutically acceptable topical carrier. In one embodiment, the aqueous solvent is present and is water, saline, growth medium (e.g., microbial culture medium or cell culture medium), or a combination thereof. In a further embodiment, both an aqueous solvent and a pharmaceutically acceptable topical carrier are present in the topical composition. In yet a further embodiment, the topical composition comprises at least one cellulose derivative.

In one embodiment, the composition comprises bacterial culture media such as Todd Hewitt medium as an aqueous solvent. In one embodiment, the aqueous solvent is present in a concentration of about 1% (w/w) to about 25% (w/w). In a further embodiment, the aqueous solvent is about 2% (w/w) to 5% (w/w) of the composition.

In one embodiment, the composition is a liquid solution. In another embodiment, the composition is a gel. In another embodiment, the composition is a solid or semi-solid foam, wax, cream or lotion.

In one embodiment, the composition comprises one of the formulations provided in Table 1. The vegetable oil, in one embodiment, is palm, olive or corn vegetable oil. It should be noted that Table 1 is simply an example of the composition components and concentrations that can be used with the present invention.

In one aspect, the present disclosure provides a method of treating a microbial infection in a subject in need thereof. The microbial infection, in one embodiment, is a bacterial, viral or fungal infection, or a combination thereof.

Without wishing to be bound by any theory, the topical GML compositions described herein are less irritating than currently approved antimicrobial compositions, resulting in a more favorable patient compliance rate, compared to other antimicrobial compositions currently used in The technique.

In one embodiment, the method comprises administering to the subject a topical composition comprising GML as described herein. In one embodiment, the method comprises topically administering to the subject an effective amount of a composition comprising GML, a vegetable oil and a pharmaceutically acceptable topical carrier. In another embodiment, the method comprises topically administering an effective amount of a composition comprising GML, a non-aqueous gel and a pharmaceutically acceptable topical carrier. In yet another embodiment, the method comprises administering to the subject one of the compositions provided in Table 1.

In one embodiment, the method of treating a microbial infection comprises applying an effective amount of one or more of the GML compositions described herein to at least one skin or mucosa surface of a subject.

In some embodiments, the composition is applied or impregnated onto a wipe, sponge, swab or other material, and then applied to the skin or mucosal surface of the subject using the respective material. As used herein, the term "swab" refers to a material suitable for applying a liquid, gel, wax, cream or lotion to a skin or mucosal surface, or to the act of applying a liquid, gel, wax, cream or lotion to the skin or mucosal surface, or the act of picking up a liquid, gel, wax, cream, lotion or fluid from the skin or mucosal surface. In some embodiments, the material is attached to a support, for example a rod, wire, rod or applicator. In further embodiments, the material attached to a support is attached to one or both ends thereof. In some embodiments, the wipe, sponge, swab or other material is preloaded or packaged along with the composition.

Certain bacteria have been shown to be resistant to the antibacterial effect of GML. Such bacteria include those with a dense layer of LPS, e.g. e.g., Enterobacteriaceae species, e.g., E. coli, as well as Pseudomonas aeruginosa. Additionally, the antimicrobial activity of GML can be inhibited by the production of lipases or other hydrolyzed enzymes such as GEH esterase, which is produced by S.aureus.

Without wishing to be bound by theory, GML is believed to inhibit microbial infection through one or more of several mechanisms including, but not limited to, direct microbial toxicity; inhibition of the entry of the infectious microorganism into the vertebrate cell; inhibition of microorganism growth; inhibition of the production or activity of virulence factors such as toxins; stabilization of vertebrate cells; or inhibition of the induction of inflammatory mediators or immunostimulators that otherwise potentiate the infectious process.

Bacteria use two-component signal transduction systems to respond and adapt to environmental changes, as well as to produce virulence factors. Without wishing to be bound by theory, GML is believed to interfere with bacterial signal transduction, either directly or indirectly, through interaction with bacterial plasma membranes. In one embodiment, the bactericidal effect of GML is mediated at least in part by interactions at the bacterial plasma membrane. In a further embodiment, GML can be detected in association with the bacterial plasma membrane, but cannot be detected in association with the cytoplasm.

In one embodiment, GML-mediated direct disruption of bacterial membranes includes interference with the localization of signaling proteins within the membrane, or interference with ligand binding to signaling proteins. In one embodiment, GML has an indirect effect on a two-component signal transduction system and the effect is selected from modifications in membrane structure that interfere with the ability of transmembrane proteins to perform signaling functions; dissipation of bacterial plasma membrane potential; and alterations of pH gradients across membranes.

In one embodiment, the indirect effect described above is mediated through one or more tetramic acids, for example those produced by P. aeruginosa and certain strains of lactobacillus. Tetramic acids produced by these organisms contain a 2,4-pyrrolidinedione ring and a side chain of 12 carbon atoms. Its properties include broad-spectrum antibacterial effects and anti-inflammatory activities. Without wishing to be limited to any theory, the mechanistic similarities between tetramic acids and GML may explain why P. aeruginosa and lactobacilli are highly resistant to the antimicrobial activities of GML. To P. aeruginosa,tetramic acids are important for the homoserine lactone quorum sensing system. For example,P. aeruginosa cultured in the presence of high concentrations of GML (>2000 μg/mL) at pH 7.0 appears to have upregulated production of numerous virulence factors, including pigments, consistent with effects associated with activation of the quorum sensing system. .

Exemplary two-component systems found in S.aureus include the agr and WalK/R regulatory system. GML has been shown to affect the agr regulatory system, which regulates several virulence factors in S.aureus.WalK/R is essential for microbial viability. Without wishing to be bound by theory, one or more two-component systems critical for microbial viability, such as, for example, WalK/R, can be directly inhibited by GML at higher doses, resulting in rapid death of microbes. .

Similar to the putative effects of GML on bacterial plasma membranes, GML has been shown to inactivate certain viruses by disrupting viral lipid envelopes [Thormar et al. (1994) Ann NY Acad Sci 724; 465].

In one embodiment, the methods described herein are used to treat a patient with a vaginal microbial infection. In a further embodiment, the vaginal microbial infection is vulvovaginal candidiasis (VVC) or bacterial vaginosis (BV). Women with BV are at risk for pelvic inflammatory disease, endometritis, and vaginal cuff cellulitis, and pregnant women with BV are at increased risk for low birth weight, preterm birth, and chorioamnionitis. In patients with VVC or BV, the vaginal flora, which is normally dominated by Lactobacillus species, is altered in such a way that other bacterial and/or fungal species dominate. Gardnerella vaginalis and other anaerobic bacteria are commonly associated with BV; Candida species, usually C.albicans, are associated with VVC. Accordingly, in one embodiment, the methods provided herein are used to treat a patient with an anaerobic bacterial infection. In a further embodiment, the infection is a Gardnerella vaginalisoCandida infection (e.g., C. albicans).

The GML compositions provided herein, in one embodiment, are used in methods of inhibiting toxin production. For example, in one embodiment, a method is provided for inhibiting a bacterial toxin and/or reducing a disease associated with a bacterial toxin. In a further embodiment, the method comprises applying one or more of the topical compositions described herein to a tampon or wound dressing, which is subsequently used by the subject or applied to the subject. In a further embodiment, the bacterial disease treated by the methods described herein is toxic shock syndrome (TSS), which is caused by the production of TSS toxin 1 (TSST-1) or, more rarely, other toxins such as enterotoxin A, B, and C, due to S.aureus. Symptoms and sequelae of TSS may include acute fever, rash, hypotension, malaise, multiple organ failure, coma, peeling skin, or death. Most cases of TSS are associated with the use of tampons during menstruation, although TSS can occur in any individual with a S.aureus infection, particularly in an individual with a skin break.

Urinary tract infections (UTIs) are particularly common in women and the elderly. UTIs usually begin in the lower urinary tract (i.e., urethra and bladder) and are usually treated with antibiotics after symptoms appear. If left untreated, a UTI can spread to the kidney and cause permanent kidney damage. UTIs are usually caused by Escherichia coli, but can also be caused by other Enterobacteriaceae, Staphylococcus aureus, other gram-positive bacteria, or, more rarely, viruses or fungal species. In one embodiment, the methods described herein are used to treat a subject having a urinary tract infection. The method comprises, in one embodiment, topically applying to a skin or mucosal surface of the patient one or more of the compositions described herein. In a further embodiment, the patient has undergone long-term antibiotic therapy prior to topical application of the composition.

To establish infection in a host subject, viruses such as HIV and SIV are believed to require an initial inflammatory response that results in the recruitment of CD4+ T cells, which are subsequently infected by the virus, to the site of infection. . In one embodiment, the methods described herein are used to treat HIV and/or SIV infections. The method comprises, in one embodiment, topically applying to a skin or mucosal surface of the patient one or more of the compositions described herein. In a further embodiment, the composition is administered intravaginally.

It is estimated that there are more than 500,000 cases of post-surgical infections with S.aureus in the United States annually, and it has been shown that 80% of these infections result from the same bacteria found in patients' anterior nasal passages. Additionally, there have been significant outbreaks of streptococcal pharyngitis and streptococcal toxic shock syndrome associated with upper respiratory tract infection with Streptococcus pyogenes, for example, an outbreak of the M3 strain. These facts suggest that nasal decolonization, when combined with possible decolonization of other parts of the upper respiratory tract and the use of surgical scrubs that kill pathogens at surgical sites, may be effective in preventing and treating postsurgical and other infections of the upper respiratory tract. respiratory tract. In some embodiments, the GML compositions provided herein are used in methods to decolonize the respiratory tract, other mucosal surfaces, or surgical incision sites to reduce strep throat, streptococcal toxic shock syndrome, or postsurgical S infections. aureus. In some embodiments, the method comprises applying one or more of the compositions provided herein to the anterior nares of a subject. For example, in one embodiment, 1 mg/mL GML in a 10% non-aqueous gel is applied to a swab and the swab is rotated around each nostril to the nasal bone 3 times.

Oral streptococci have been reported to be involved in gum disease and dental caries and, in susceptible individuals, infective endocarditis. The GML compositions provided herein are used in some embodiments to prevent or treat streptococcal infections that cause dental caries, gum disease, and infective endocarditis. The method in one embodiment comprises applying to the teeth and gum lines of a subject one or more of the compositions provided herein. For example, in one embodiment, 1 mg/mL GML in a 5% non-aqueous gel is applied to the teeth and gum lines of a subject using a swab.

In some embodiments, the subject has a bacterial infection. Bacterial infections that can be treated with the topical compositions provided herein include, but are not limited to, infections caused by the following bacteria: Staphylococci (e.g., S.aureus, S. intermedius, S. epidermidis) , Group A Streptococcus (e.g., S.pyogenes), Group B Streptococcus (e.g., S.agalacticae), Group C, F, and G Streptococcus, Streptococcus pneumoniae, Bacillus anthracis, Peptostreptococcus species, Clostridium perfringes , Neisseriae gonorrheae, Chlamydia trachomatis, Haemophilus influenzae, Pseudomonas aeruginosa, Helicobacter pylori, Gardnerella vaginalis, Bacteriodes fragilis, Burkholderia cepacia, Bordatella bronchiseptica, Campylobacter jejuni, Enterobacteriacae (e.g., Escherichia coli), Pasteurella multocida, and Mycobacterium (e.g. e.g., M. tuberculosis and M. phlei).

Furthermore, the topical compositions described herein, in one embodiment, are used to treat one or more bacterial infections caused by one or more of the bacteria listed in Table 2. Table 2 shows the results of experiments testing the activity GML's antibacterial action against various bacteria grown under optimal growth conditions. Burkholderia cenocepacia, which used to be called Pseudomonas cepacia and is related to Pseudomonas aeruginosa, was killed by GML at concentrations of 500 μg/mL. Mycobacterial species typically produce large amounts of complex fatty acids. However, these organisms were killed by GML at concentrations of >50 μg/mL. In addition to inhibiting the growth of gram-positive bacteria, GML inhibited the production of exotoxins independently of the inhibition of the growth of all the organisms analyzed (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcide groups C, F, and G, and Clostridium perfringens The most susceptible organisms to be killed by GML were species of Peptostreptococcus, Clostridium perfringens, Bordetella bronchiseptica, and Campylobacter jejuni, all of which were killed by GML (1 μg/mL).

In some embodiments, the subject to be treated with one or more of the topical compositions provided herein has a viral infection. In a further embodiment, the viral infection is caused by one or more of the following viruses or class of viruses: influenza virus, herpesvirus (e.g., herpes simplex virus 2), lentivirus (e.g., herpes simplex virus 2), of human immunodeficiency).

In some embodiments, the subject to be treated with one or more of the topical compositions provided herein has a fungal infection. In a further embodiment, the fungal infection is caused by one or more of the following species of organisms: Candida species (e.g., C. albicans), Microsporum species, Trichophyton species, Epidermophyton floccosum, Penicillium species, Aspergillus species, Trichomonas vaginalis.

Methods for identifying and diagnosing a bacterial, viral or fungal infection are generally known to those skilled in the art. To evaluate whether the formulations described herein are useful for treating an infection, methods known to those skilled in the art can be employed. For example, a BV infection before and after treatment can be evaluated by microscopic examination of vaginal cells.

In one embodiment, a method is provided for removing or killing a biofilm comprising one or more microorganisms. Biofilms may be implicated in UTIs, oral infections, and dental diseases such as gingivitis, and may also form on the surface of implanted devices, including prostheses, catheters, or heart valves. In one embodiment, the method comprises administering the topical composition by applying it directly to the biofilm.

In some embodiments, the methods of the disclosure comprise administering a second active agent, together with GML or a derivative of GML. The additional active agent may be present in the compositions described herein or may be administered separately. In one embodiment, one or more additional active agents are administered before or after the topical GML composition. For example, the two active agents may be administered topically in series or administered serially by different routes of administration.

In one embodiment, the additional active agent(s) are administered before, during or after administration of the composition of the disclosure. In another embodiment, the additional active agent(s) are administered by the same route as the composition or by a different route. For example, the additional active agent(s), in one embodiment, are administered via one of the following routes of administration: topical, intranasal, intradermal, intravenous, intramuscular, oral, vaginal , rectal, otic, ophthalmic, subcutaneous. The dosage of additional active agents depends, for example, on the nature of the infection or disease, the site of administration, the weight, age, sex and surface area of the subject, concomitant medications; and medical judgment.

Additional active agents include, for example, antibiotics, antiviral agents and antifungal agents. Antibiotics include, without limitation, aminoglycosides, carbacephemes, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, penicillins, polypeptides, quinolones, sulfuramides and tetracyclines. Antifungal agents include, without limitation, those of the azole, polyene or echinocyanin class, nucleoside analogues, allylamines, griseofulvin, tolnaftate or selenium compounds. Antiviral agents include, for example and without limitation, acyclovir, ganciclovir, valganciclovir, abacavir, enofovir, lamivudine, emtricitabine, zidovudine, tenofovir, efavirenz, raltegravir, enfuvirmide, maraviroc, ribavirin, amantadine, rimantadine, interferon, oseltamivir and zanamivir.

Examples

The present invention is best illustrated with reference to the following examples. These examples, like the embodiments described above, are illustrative of the invention.

Example 1: Antimicrobial effects of GML in vegetable oil

A study was carried out to evaluate the ability of various concentrations of GML in olive, palm or corn oil to inhibit the growth of various bacterial or fungal microorganisms in vitro.

GML was preheated in olive (Figure 1), palm (Figure 2), or corn (Figure 3) oil at 37 °C to melt the GML and then added to 1 mL of Todd Hewitt broth (Difco, Detroit MI) in tubes. round bottom in concentrations ranging from 10 μg/mL to 5000 μg/mL. The following microbes were added to the tubes at the indicated concentrations: Staphylococcus aureusMNPE (methicillin sensitive, 1x107/mL); Staphylococcus aureusMW2 (methicillin resistant, 1x107/mL); mL); oCandida albicans(1x106).

The tubes were shaken at 37°C at 200 revolutions per minute (RPM) in standard air for 18 hours. Plate counts were performed to determine colony forming units/mL (CFU/mL).

In the presence of only 10 μg/mL of GML, the CFU/mL of G.vaginalisse was reduced in the three vegetable oils tested; The growth of both G.vaginalis and S.agalactia was completely or almost completely inhibited at levels of 20 μg/mL GML or higher in the 3 vegetable oils tested. Furthermore, the growth of C.albicans and both S.aureus strains was inhibited at 100 μg/mL of GML and the growth was completely inhibited by GML at 500 μg/mL and 5000 μg/mL of GML, in the 3 vegetable oils tested. Therefore, GML was effectively antimicrobial when mixed with olive, palm or corn oil.

Example 2: Effect of GML on microorganisms growing as biofilms

To evaluate the effect of GML on biofilms, S.aureus biofilms were cultured. S.aureus128, an organism that expresses toxic shock syndrome toxin-1 (TSST-1), was inoculated at a rate of 107/0.1. mL inside the previously moistened dialysis tubing that had been tied at one end. Then, a buffer was inserted into the dialysis tube and the tube was immersed in Todd Hewitt broth (Difco Laboratories, Detroit, MI) containing 0.8% agar. The open end of the dialysis tube remained above the surface of the agar, so that the only source of nutrients for the growing microbes was the medium absorbed through the dialysis tube. Figure 4 shows biofilm growth on buffer fibers and cellulose acetate dialysis tubes after an 18-hour incubation, at magnifications of 6000x (A) and 9000x (B).

The buffer bags were incubated on solidified agar. At 4, 8, 12, and 16 hours, buffer sacs were removed from the agar, sliced open, and weighed to determine fluid gain. TSST-1 was eluted by the addition of phosphate-buffered saline (PBS). The cumulative amount of TSST-1 was quantified by first concentrating the eluted fluids by adding 4 volumes of absolute ethanol, then resolubilizing them in distilled water, and analyzing them by Western immunoblotting. Figure 5 shows the increasing amounts of TSST-1 present in the buffers over time. TSST-1 was not detected in the presence of 5% GML (data not shown).

To directly evaluate the effect of GML on biofilm formation, 96-well plastic microtiter plates were inoculated with approximately 106/mL of one of three S.aureus strains (MN8, a methicillin-sensitive strain; MNWH, a methicillin-resistant strain; or MW2, a methicillin-resistant strain), or with nontypeable Haemophilus influenzae strains. Wells were cultured stationary at 37°C for 24 and 48 hours (Figures 6-8). As a control, in a set of three wells for each microbe, the wells were shaken 3 times by pipetting up and down. The bactericidal activity of GML was determined by measuring CFU/mL in supernatants. After removing the supernatants, the wells were washed three times with PBS to remove unattached cells and then treated with crystal violet for 30 min. The wells were washed again three times with PBS to remove unbound crystal violet. Finally, the wells were treated with ethanol to solubilize the crystal violet associated with the biofilms. Absorbances at 595 nm were determined using an ELISA reader to measure biofilm formation.

Figures 6, 7 and 8 provide the results of the study. The growth of all three S.aureus strains was completely inhibited by GML at 500 μg/mL at both 24 and 48 hours, as measured by CFU/mL (Figure 6; dashed line indicates initial inoculum size ; * significant reduction in the mean CFU/mL compared to the initial inoculum, p< 0.001). In contrast, at GML concentrations 10-fold lower than those needed to inhibit bacterial growth, biofilm formation was significantly inhibited as measured by reduction in crystal violet staining of biofilm material retained in the wells of the plates. microtiter (Figure 7; # significant reduction in mean absorbance at 595 nm compared to wells without GML, p<0.01).

Similarly, GML was bactericidal against H. typeable influenzaeno in the context of a biofilm at concentrations of 50 μg/mL (Figure 8, top; dashed line indicates initial inoculum size; *significant reduction in mean CFU/mL compared to initial inoculum, p< 0.001). Furthermore, GML inhibited the biofilm formation of H. influenza at concentrations as low as 1.0 μg/mL, measured at 24 and 48 hours (Figure 8, bottom; # significant reduction in mean absorbance at 595 nm compared to no GML control, p<0.01).

To evaluate the effect of GML on previously formed biofilms, side-by-side wells that were not treated with GML throughout the incubation and that had high absorbances at 595 nm at 48 hours were used, indicating that a biofilm in the well, were treated with 500 μg/mL of GML for 60 minutes at 37 °C. Supernatants were removed and bactericidal activity was determined by measuring CFU/mL (Figure 9, left; # significant reduction in mean absorbance at 595 nm compared to no GML control, p<0.01). The wells were then washed and stained with crystal violet as described above.

Figure 9 provides the results of the study. After 48 hours, 500 μg/mL GML killed both S.aureus and H. influenzae (Figure 9, left). Similarly, 500 μg/mL GML almost completely removed biofilm material attached to the wells, as demonstrated by the loss of crystal violet staining in GML-treated wells (Figure 9, right).

Example 3: Synergistic effects of GML and EDTA on the growth of E. CoI!

Previous studies indicated that Enterobacteriaceaetal species such as E. coli, as well as Pseudomonas aeruginosa, were resistant to the antibacterial effects of GML and suggested that the dense LPS layer protected against GML in these organisms. An additional study was conducted to determine whether the addition of EDTA to a GML composition would improve the antimicrobial properties of the GML composition.

E. coli (Watson strain) was adjusted to 1.2x107/mL in Todd Hewitt medium. Various concentrations of EDTA ranging from 0.05 M to 0.00005 M were added to the wells, in the presence or absence of 100 μg/mL GML. Cultures were incubated with shaking (200 RPM) for 24 hours, at which time samples were removed for plate counting.

The results of the study are shown in Figure 10. EDTA alone inhibited the growth of E. colialgo, in a dose-dependent manner. The combination of 100 μg/mL GML with EDTA showed higher antibacterial activity (*p<0.001; the dashed line indicates the initial inoculum size).

To further evaluate the combined effects of GML and EDTA, the experiment was carried out as above, and the relative effects of EDTA alone on the growth of E. colio combination of GML and EDTA were evaluated in direct comparison with GML alone. The results are given in Figure 11. As in the experiment described above, EDTA alone inhibited the growth of E. coli to some extent at higher concentrations, while the combination of 100 μg/mL GML with EDTA showed higher antibacterial activity, in a dose-dependent manner. GML alone did not exhibit any bactericidal activity against E. coli.Therefore, GML and EDTA exerted a synergistic antibacterial effect.

Example 4: The effect of pH on the activity of GML against pathogenic microorganisms

Because the presence of EDTA made aE. colisusceptible to GML, it was hypothesized that surface protonation of E. colioP. aeruginosa may increase GML activity by repelling divalent cations, thereby altering the integrity of LPS.

An experiment was performed to determine the effect of GML in the presence of various pH levels on the growth of E. coli. E. was grown. coli (Watson strain) in Todd Hewitt medium and was adjusted to 107 CFU/mL. Using acetate buffer, the pH of the cultures was adjusted to 5.0, 6.0, or 7.0. GML was added to the cultures at concentrations of 5000, 50 or 0.1 μg/mL and the cultures were incubated at 37 °C with shaking (200 RPM). Samples were removed for CFU/mL counting after 24 hours.

The results of the study are provided in Figure 12.E. colino was susceptible to GML at pH 7.0, even when up to 5000 μg/mL GML was added to the culture. However,E. coli was very susceptible to GML at pH 6.0, since GML at 50 μg/mL was bactericidal, and was even more susceptible to GML at pH 5.0, since GML only at 0.1 μg/mL was bactericidal. in these crops. With each unit of decrease in pH,E. coli appeared to become 500 times more susceptible to GML (*p<0.001; dashed line indicates initial inoculum size).

A similar experiment was carried out to evaluate the effect of GML on the growth of Haemophilus influenzaea at various pHs. 1 μg/mL GML had no effect on the growth of H. influenzaea a pH of 7.0 (Figure 13). However, at pH 6.0, 1 μg/mL GML completely abrogated the growth of H. influenzaea 4, 8 and 24 hours (Figure 13).

The effect of GML in a range of concentrations and in the presence of a range of pH levels on the growth of Pseudomonas aeruginosa was also determined. P. was inoculated. aeruginosa (strain PA01) in Todd Hewitt broth at 5.7x106/mL. GML was added to the cultures at a concentration range of 10 μg/mL to 5000 μg/mL and the pH was adjusted to 5.0, 6.0, or 7.0. CFU/mL were determined after 24 hours of incubation. The results of the study are shown in Figure 14. At pH 6.0 or 7.0, no concentration of GML inhibited the growth of P. aeruginosa. A pH of 5.0 in the absence of GML was somewhat inhibitory for the growth of P. aeruginosa. However, the addition of GML to the cultures at pH 5.0 further inhibited the growth of P. aeruginosa in a dose-dependent manner (*p<0.001; the dashed line indicates the initial inoculum size).

The results of the study indicated that GML and reduced pH synergistically inhibited the growth of pathogenic microorganisms.

Example 5: Effect of non-aqueous gel on the antimicrobial activity of GML

Non-aqueous gels composed of propylene glycol (73.55% w/w), polyethylene glycol 400 (25% w/w), and hydroxypropyl cellulose (Gallipot, St Paul, MN; 1.25% w/w) were heated to 65 °C. with or without GML. After solubilization of the components, the gels were diluted with 10% or 25% Todd Hewitt broth. GML was also diluted only comparably with Todd Hewitt broth to serve as an additional control. S.aureus (strain MN8, a strain with toxic shock syndrome) was incubated at 37 °C with shaking (200 RPM) in various concentrations of nonaqueous gel in the presence of GML at concentrations ranging from 1 μg/mL to 5000 μg/mL or in the presence of GML only at the same concentrations. After 24 hours, plate counts (CFU/mL) were determined.

Figure 15 shows the results of the study. GML inhibited the growth of S.aureusa at a concentration of 100 μg/mL or higher. The 10% and 25% nonaqueous (NA) gel concentrations, as well as the range of GML concentrations, exhibited dose-dependent effects on the growth of S.aureus. The 25% nonaqueous gel GML had approximately 500 times greater activity than GML alone, and GML in 10% non-aqueous delivery vehicle had approximately 10 times greater activity than GML alone (Figure 15; *p<0.001; dashed line indicates initial inoculum size). Therefore, the combination of the non-aqueous gel and GML had a potent antimicrobial effect.

Example 6: Solubility of tenofovir in GML gels

Non-aqueous gels composed of propylene glycol (73.55% w/w), polyethylene glycol 400 (25% w/w) and hydroxypropylcellulose (1.25% w/w) were prepared in a pH range of 4.0 to 4. 5. The anti-HIV drug tenofovir was added to the gels at a concentration of 10 mg/mL to determine if the drug was soluble in the compositions.

The results of the study are shown in Figure 16. Tenofovir (10 mg/mL) was not soluble in the non-aqueous gel at pH 4.0 - 4.3, but was soluble in the non-aqueous gel at pH 4.4 or 4.5.

Example 7: Effectiveness of various forms of GML

Multiple forms of GML exist, including R or S optical isomers and GML with lauric acid ester attached at the 1/3 or 2 positions of glycerol. To test potential differences between the optical isomers, the R form of GML, which is commercially available, was compared with a racemic mixture of GML from the R and S forms. To test possible differences between GML with lauric acid attached to different glycerol positions , GML bound at position 2 was compared with that of commercially available purified lauric acid with a mixture of single GML with lactic acid at positions 2 and 1/3. The antibacterial effects of these forms of GML were evaluated in S.pyogenes (strain 594).

The results of the study are shown in Figure 17. The R form of GML and the mixture of the R and S forms of GML had the same antibacterial activity (left panel; the dashed line indicates the initial inoculum size). However, the GML form with lauric acid at position 2 was 2 times more active than the mixture of GML forms (right panel; *p<0.001; dashed line indicates initial inoculum size). Therefore, the results indicated that the activity of GML was not dependent on chirality, but to some extent depended on the position of lauric acid.

Example 8: Antibacterial activity of GML versus lauric acid

The bactericidal activity, as well as the ability to inhibit exotoxin production, of GML was determined in comparison with lauric acid (a major cleavage product of GML). S.aureus, an organism that produces glycerol-ester hydrolase (GEH), and S.pyogenes, which does not produce GEH, were tested.

The results of the study regarding the bactericidal activity of GML and lauric acid are shown in Figures 18 and 19. The GML and lauric acid were incubated at the indicated concentrations with approximately 5 x 106 CFU/mL of S.aureusMN8 (Figure 18) or S.pyogenes (Figure 19) for 24 hours at 37 °C, in triplicate. For S.aureus, plates were incubated with shaking (200 RPM). For S.pyogenes, the plates were incubated stationary in the presence of 7% CO<2>. Plate counts were used to determine CFU/mL. Bactericidal activity was defined as the minimum concentration of GML or lauric acid necessary to reduce CFU by at least 3 logs. GML was bactericidal for S.aureuse at concentrations 200 times lower than lauric acid (Figure 18; *p<0.001; dashed line indicates initial inoculum size). GML was bactericidal for S.pyogenesa at concentrations 500 times lower than lauric acid (Figure 19; *p<0.001; dashed line indicates initial inoculum size). Furthermore, when comparing the bactericidal activity of GML against the two organisms, GML was 5 times more bactericidal for S.pyogen than for S.aureus.

To determine the relative ability of GML and lauric acid to inhibit superantigen production, S.aureusMN8 and S.pyogenes were grown for 8 hours in the presence of GML or lauric acid. The production of TSST-1 by S.aureusMN8 and the production of streptococcal pyrogenic exotoxin A (SPE A) by S.pyogenesse were quantified by Western immunoblot analysis.

The results of the study regarding superantigen production are shown in Figure 20. Both GML and lauric acid significantly inhibited exotoxin production by S.aureusMN8 and S.pyogenes at concentrations that did not inhibit growth. However, the concentration of GML required to inhibit exotoxin production was lower for both organisms compared to the concentration of lauric acid required to inhibit exotoxin production. GML inhibited the production of TSST-1 by S.aureuse at a concentration of 0.2 μg/mL and inhibited the production of SPE A by S.pyogenes at a concentration of 0.025 μg/mL, while lauric acid inhibited the production of both of TSST-1 and SPE A at a concentration of 2.5 μg/mL. (*p<0.01).

To further evaluate differences between S.aureus and S.pyogenes with respect to GML activity, GML was pretreated by incubating overnight at a Todd Hewitt broth concentration of 1000 μg/0.4 mL with 0.1 mL of culture fluid. sterile stationary phase of S.aureusMN8 or S.pyogenes.

The results of the study are shown in Figure 21. Preincubation with S.aureuse eliminated the antibacterial activity of GML against S.aureusMN8 measured in the 24-hour assay described above. However, preincubation with S.pyogenes did not affect the antibacterial activity of GML (*p<0.001; the dashed line indicates the initial inoculum size). These results suggested that an esterase produced by S.aureus, such as GEH, inhibited the activity of GML.

Example 9: Development of resistance to GML in S. Aureus

To determine if S.aureus developed resistance to GML, S.aureus cultures were treated with suboptimal concentrations of GML (50 μg/mL) for one year. Each week, S.aureus strain MN8 was transferred to Todd Hewitt agar plates containing 50 μg/mL GML and cultured for 48 hours. This suboptimal concentration of GML allows S.aureus to grow for 48 hours. Growing organisms were passed weekly to fresh plates containing 50 μg/mL GML, or transferred to plates containing 100 μg/mL GML, a concentration at which S.aureus normally cannot grow for 24 hours. Additionally, 50 μg/mL GML plates were plated at 4 °C weekly to allow GML to crystallize. Plates were analyzed for non-crystallizing zones around individual S.aureus colonies, which is indicative of GML cleavage by GEH.

Although S.aureus exhibits rapid development of resistance to many antibiotics, no developed S.aureus was able to grow on 100 μg/mL GML plates. Therefore, S.aureus did not develop resistance to GML during the one-year period. Furthermore, no mutant was identified that had increased GEH production during the passage year (data not shown).

Example 10: Decolonization studies

A study was conducted to evaluate the ability of GML (5% w/v), formulated in a non-aqueous gel, to decolonize the respiratory tract in humans and to decolonize contaminated surgical incision sites in experimental rabbits.

The anterior nares of three human subjects were swabbed with swabs to assess whether GML was capable of decolonizing the respiratory tract. The swabs were immersed in phosphate-buffered saline (PBS), which had previously been shown to result in the absorption of 0.1 mL of PBS, and then used to clean the anterior nares of each subject. The swabs were rotated around each nostril to the nasal bone 3 times. Colony-forming units of microbes from swabs were determined by plate counts on blood agar and mannitol salt agar. The anterior nares were then treated in the same manner with swabs that had been dipped once in GML gel. Swabs were collected from the anterior nares of each participant at designated time periods for up to 24 hours, and the swabs were cultured to grow S.aureus and coagulase-negative staphylococci.

The data obtained from this study are shown in Figure 22. The data were log transformed before performing statistics due to the high variability in the CFU present in the three subjects. The right and left anterior nares of all three subjects contained an average log CFU/mL of S.aureus of 1.6 or 1.5, respectively, before treatment with<g>M<l>. GML treatment significantly reduced (p<0.05 by Student's t-test analysis) S.aureuse counts in both nostrils to 0 CFU/mL at all time points tested after treatment, including the 24-hour time point. hours. The right and left anterior nares of all three subjects contained an average log CFU/mL of coagulase-negative staphylococci of 3.9 and 3.8, respectively, before GML treatment. GML treatment significantly (p<0.05) reduced coagulase-negative staphylococcus counts in both nostrils at analyzed time points of 4 hours or more after treatment, including the 24-hour time point.

For one subject, the persistence of reduced CFU of both S.aureus and coagulase-negative staphylococci was analyzed for 3 days. S.aureus counts remained at 0 throughout the 3-day analysis period. CFU of coagulase-negative staphylococci also remained low throughout the period (initially there were 560 and 880 CFU/mL in the right and left nares, respectively; after 3 days, 8 and 0 CFU/mL of coagulase-negative staphylococci were detected in the right and left nostrils, respectively; data not shown).

Next, studies were conducted to evaluate whether the 5% GML gel could decolonize teeth from oral aerobic bacteria. Human volunteers were sampled with a swab saturated with PBS along the teeth and gum lines on the left side of the mouth and analyzed for CFU/mL of total bacteria subsequently cultured on blood agar plates. The same individuals were then rubbed with GML gel by running the gel over the teeth and gum lines on the right side of the mouth. Enough gel was used to cover the entire surface of the teeth and gums. Thirty minutes after treatment, participants were sampled with PBS-saturated swabs on the right side and total CFU/mL were determined.

To ensure that the data obtained did not differ simply due to the removal of bacteria by the initial swab rub, different sides of the teeth and gum lines were used for the pre- and post-treatment swab rubs. The bacterial counts on both sides of the teeth and on the gums were assumed to be approximately equal, and a pre-test swab rub confirmed that to be the case.

The results of the study are shown in Figure 23. The data were log transformed to account for the high variability in CFU/mL between subjects. There was a >5 log reduction in CFU between pretreatment swabs and posttreatment swabs, indicating that GML killed bacteria attached to the teeth, which were primarily oral streptococci. Since GML was effective in reducing counts, the data also suggested that the gel with GML was effective in removing and/or killing bacteria in biofilms, which would be expected to be present on teeth. The data were very significantly different as analyzed by Student's t-test analysis (p<0.001).

In a final study, S.aureus strain MN8 (1 x 1010 CFU) was used to coat the surgical incision sites of three rabbits per group. The surgical incision sites were 4 cm subcutaneous incisions that had been closed with 4 silk sutures (Ethicon, Cornelia, GA). After closure, the incision sites of three animals were rubbed with 5% w/v GML non-aqueous gel swabs, and the incision sites of control animals were rubbed with PBS swabs. Enough gel was rubbed with GML to provide a uniform coating of the surface. After 24 hours, rabbits were examined for inflammation (determined by redness at the incision sites) and total CFU/mL that could be obtained by swabbing the incision sites with swabs saturated with PBS.

The results of the study are shown in Figures 24 and 25. Rabbits that were swabbed with PBS had an average of 8.8 log CFU/mL of S.aureusa over 24 hours (Figure 24) and obvious inflammation at the site. surgical (Figure 25). In contrast, in rabbits that had been treated with GML, no CFU were detected at 24 hours (Figure 24; p<<0.001 by Student's t-test analysis of log-transformed data) and there was less inflammation at the site. surgical (Figure 25).

Taken together, the data presented in this study showed that a non-aqueous 5% GML gel could be used effectively to reduce colonization by potential pathogens of the nasal and oral cavities of humans and surgical incision sites in rabbits.

Example 11: Clinical efficacy of GML in vaginal infection

The following predictive example provides a proposed clinical study in which the relative efficacy of a composition comprising GML, a vegetable oil, and a pharmaceutically acceptable topical carrier will be determined for the treatment of BV or VVC. This prophetic example is intended to illustrate the principles of the present invention.

The study design is a double-blind, single-center, 60-subject study, 20 subjects per arm. Subjects will be stratified by type of vaginal infection (BV, VVC, or both) and age.

Study subjects will be women between the ages of 18 and 50 with BV or VVC (as determined by the gynecological examination performed during the screening visit) who have signed informed consent. Pregnant women, menstruating women, and women who have had a systemic infection or used a vaginal antimicrobial, anti-inflammatory, or immunosuppressive medication in the previous 4 weeks will be excluded.

Study Endpoints and Treatment Evaluation: A vaginal swab will be collected at the baseline visit. Subjects will self-administer one of the following preparations vaginally every 12 hours for two days, for a total of 4 doses: 0% (vehicle control), 0.5% GML or 5% in olive oil. Vaginal swabs will be collected 12 and 48 hours after the final dose of study drug or vehicle control. Colony Forming Units (CFU) will be determined for Lactobacillus, G. vaginalis, and Candida in initial and treatment follow-up vaginal swabs. Subjects will be followed over a period of 3 months and all adverse events including clinical findings and opportunistic infections will be recorded.

The results of the proposed study will be used in the development of a clinical protocol for the treatment of BV or VVC.

Example 12: Clinical use of GML in urinary tract infection

The following prophetic example is intended to illustrate circumstances in which the formulations described in this document are indicated.

A subject at risk for urinary tract infections (e.g., a woman or an elderly individual) can apply a composition comprising 50 μg/mL of GML to a sponge, and then apply the sponge to the area of the opening. or external urethral orifice. The composition can be applied once a day to prevent urinary tract infections. The composition may further comprise an accelerant such as EDTA and/or a non-aqueous gel.

Example 13: Clinical use of GML in cellulite

The following prophetic example is intended to illustrate circumstances in which the formulations described in this document are indicated.

A subject who has been diagnosed with cellulitis can topically self-administer a composition comprising 5 μg/mL GML and 25% non-aqueous gel in a pharmaceutically acceptable topical vehicle to the site of skin infection, twice daily until the infection resolves. If medically indicated, the patient may also be administered an antibacterial agent.

Claims (9)

1. A composition for use in the topical treatment of Gardnerella vaginalis, Haemophilus influenzae, Escherichia coli and/or Pseudomonas aeruginosa, wherein the composition comprises: R isomers, S isomers or racemic mixtures of isomers of glycerol monolaurate (GML) with lauric acid in the 1/3 positions, glycerol monolaurate (GML) with lauric acid in the 2 position, or combinations thereof, a non-aqueous gel, a vegetable oil, an accelerant and a pharmaceutically acceptable topical carrier, wherein the accelerant is an organic acid, a chelator, an antibiotic agent, an antifungal agent, an antiviral agent or a combination thereof, Wherein the pH of the composition is in the range of 4-4.5. 2. The composition for use according to claim 1, wherein the vegetable oil is palm oil, olive oil, corn oil, canola oil, coconut oil, soybean oil, wheat germ oil or a combination of the same. 3. The composition for use according to claim 1 or 2, wherein the non-aqueous gel is composed of propylene glycol, polyethylene glycol, hydroxypropyl cellulose, hydroxyethyl cellulose or a combination thereof. 4. The composition for use according to claim 3, wherein the hydroxypropyl cellulose or hydroxyethyl cellulose is present in a concentration of about 0.1% (w/w) to about 5.0% (w/w), or the propylene glycol is present in a concentration of about 65% (w/w) to about 80% (w/w) or the polyethylene glycol is present in a concentration of about 20% (w/w) to about 35% (w/w). 5. The composition for use according to claim 3, wherein the polyethylene glycol is in the molecular weight range of about 300 to about 600. 6. The composition for use according to claim 1, wherein the GML is present in the composition at about 0.001% (w/v) to about 10% (w/v) of the composition or at a concentration of about 10 μg/ mL to approximately 100 mg/mL. 7. The composition for use according to claim 1, wherein said pharmaceutically acceptable topical carrier is petroleum jelly. 8. The composition for use according to claim 1, wherein the accelerant is EDTA. 9. The composition for use according to any one of claims 1 to 9, wherein the topical treatment is carried out with a sponge, a washcloth or a swab.